This is a competitive renewal for ROI CA79976 "Dendritic cell-based genetic immunotherapy for melanoma" in which we request support for years 05-08. Our accomplishments in the previous funding period include: 1. defining the immunological events taking place in a murine melanoma model using dendritic cell (DC) engineered with a defined tumor antigen MART-1, 2. completing a phase l/II clinical trial in melanoma patients receiving MART-1/27-35 peptide pulsed DC and 3. opening a gene therapy trial using adenovirus (AdV) MART-1-transduced DC. Based on this progress, we propose to continue our translational studies of genetic immunotherapy of human melanoma centered around three specific aims. Aim 1: Genetic Immunotherapy in a CDS-Deficient Environment. We have made the remarkable and original observations that CD8 or Class I knock out mice immunized with AdVMART1-transduced DC have superior levels of protection to B16 melanoma than wild type (wt) mice. Since wt mice depleted of CD8 cells are unable to generate protective immunity, CD8 KO mice have developed a compensatory mechanism from generating robust tumor immunity to DC vaccination. We present preliminary evidence that this antitumor immunity is mediated by collaboration between effector cells of the innate (NK-like) and adaptive (CD4) arms of the immune systems. We propose to characterize the underlying mechanism. Aim 2: The Biology of Class I and Class II-Restricted T Cell Responses in AdVMART1/DC Immunized Patients with Melanoma. This clinical trial, in which patients with stage IV MART-1-positive melanoma are immunized with AdVMART1/DC, provides a unique opportunity to define immunological events triggered by genetic immunization to a defined "self" tumor antigen. Only two epitopes have been described for this small protein-HLA-A2.1-restricted MART-1/27-35 and HLA-DK4 restricted MART-1/51-73. Using ELISPOT and tetramer assays for this class I and II epitopes, we will quantitate, isolate and study MART-1-reactive CD8 and CD4 T cell in immunized patients. We will also study the role of determinant spreading and cross-presentation in clinical response, the biology of DC used for vaccination and the possible participation of innate (NK) immunity in DC-based immunotherapy. Aim 3: CTLA4 Blockade in Clinical Dendritic Cell-Based Immunotherapy. DC-based immunotherapy generates occasional but dramatic clinical antitumor responses. We have closely studied one subject in whom the administration of MART-1/DC vaccines was followed by a CTLA4 blocking antibody. Immunological analysis suggests that the antitumor immune response initiated by the DC vaccines was maintained by CTLA4 blockade. To test this hypothesis, we have designed a phase II randomized trial with the primary goal of detecting the effect of MART-1/27-35/DC + CTLA4 blockade on the frequency of melanoma antigen-specific activated T cells using ELISPOT assays. This trial will provide insight in the autoregulatory mechanisms that govern the activity of DC-based immunotherapy. In summary, we propose to continue our translational program in genetic immunotherapy with an emphasis on immune mechanism and clinical hypothesis-testing.